Vitamin B6 is an essential coenzyme, required by nearly all enzymes involved in amino acid metabolism. Studies of the biosynthesis of vitamin B6 in Escherichia coli have identified the enzymes involved, and their functions. PdxJ, or pyridoxine 5'-phosphate synthase, is known to catalyze the condensation of 1 -aminoacetone-3-phosphate and 1 -deoxy-D-xylulose-5-phosphate to form pyridoxine 5'-phosphate, the precursor to the active form of vitamin B6. However, the precise mechanism of cyclization is not well understood. A two-fold approach is proposed to probe the mechanism of pyridoxine 5'-phosphate synthase. First, site-directed mutagenesis will be used to investigate several active site residues to determine their roles in catalysis and/or in the binding of substrates. Second, rapid quench studies will be carried out to trap intermediates that would not normally be released from the active site. Isolation and characterization of these intermediates is expected to delineate the detailed mechanistic pathway from 1-aminoacetone-3-phosphate and 1-deoxy-D-xylulose to pyridoxine 5-phosphate.